CN103382229A - Preparation method and structural identification for novel strongylocentrotus nudus egg polysaccharide having immunoregulation effect - Google Patents
Preparation method and structural identification for novel strongylocentrotus nudus egg polysaccharide having immunoregulation effect Download PDFInfo
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- CN103382229A CN103382229A CN2013102481868A CN201310248186A CN103382229A CN 103382229 A CN103382229 A CN 103382229A CN 2013102481868 A CN2013102481868 A CN 2013102481868A CN 201310248186 A CN201310248186 A CN 201310248186A CN 103382229 A CN103382229 A CN 103382229A
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- polysaccharide
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- strongylocentrotus
- gonad
- egg
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- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention particularly provides a strongylocentrotus nudus egg polysaccharide with an alpha-1,4 glycosidic bond backbone and an alpha-1,3 glycosidic bond side chain, and a preparation method and an application thereof, and belongs to the natural polymer field. The invention provides the method for extracting the polysaccharide from a gonad of Yantai strongylocentrotus nudus, a chemical structure and the application of the polysaccharide. Chemical analysis and magnetic resonance spectroscopy are utilized to analyze the structure, a repeating structural unit of the strongylocentrotus nudus egg polysaccharide is found to contain 1,3 glycosidic bonds and 1,4 glycosidic bonds, wherein the backbone is composed of the 1,4 glycosidic bonds, the side chain is composed of the 1,3 glycosidic bonds, sugar residues are in an alpha configuration, the relative molecular weight is 6.78*10<5> Da, the specific rotation is [alpha]D<20>+57 DEG (c0.1, H2O), and the polysaccharide is a novel-structured glucan separated from the gonad of the strongylocentrotus nudus (strongylocentrotus nudus eggs) for the first time. At the same time, the strongylocentrotus nudus egg polysaccharide can stimulate the proliferation of spleen lymphocytes and macrophages of mice and can also enhance an ability of macrophage phagocytosis of neutral red, so that the strongylocentrotus nudus egg polysaccharide is indicated to have the function of immunological enhancement.
Description
Technical field
The present invention is specifically related to SEP-1 of a kind of α of having-Isosorbide-5-Nitrae glycosidic link main chain and α-1,3 glycosidic link side chain and its production and use; Belong to the natural polymer field.
Background technology
The sexual gland of female sea urchin is that gonad of Hemicentrotus seu Strongylocentrotus is famous and precious marine products treasure, is called as " sea urchin roe paste ", and is expensive on the world market, and the title of " byes " is arranged.Gonad of Hemicentrotus seu Strongylocentrotus contains rich in protein, polysaccharide, lipid acid, the multiple nutritive ingredients useful to human body such as VITAMIN and various trace elements.Therefore gonad of Hemicentrotus seu Strongylocentrotus not only has higher edibleness, also has simultaneously fabulous pharmaceutical use, is especially strengthening body immunity, and there is effect preferably the prevention and cure of cardiovascular disease aspect.The method of using the various pyogenic infections of sea urchin treatment is early on the books in traditional medicine.Along with the progress of science and technology, the antitumor action of sea urchin has obtained affirming of current medical science.
Abroad the research of sea urchin started from the sixties in last century, early stage research mainly concentrates on giving birth in sea urchin spinochrome and sea urchin or the research of the fungal component meta-bolites aspect.1981, George separated from poisonous lytechinus variegatus (S.droebachiensis) first and obtains two kinds of glycoprotein with anti-tumor activity.Begin since then, increasing scholar is from the shell of different types of sea urchin, internal organ, found in the sources such as ovum multiple have antitumor, the different bioactive polysaccharide such as immunomodulatory, glycoprotein, the compounds such as glycolipid.
China be the sea urchin resource than more rich country, and polysaccharide is nutritive ingredient important in gonad of Hemicentrotus seu Strongylocentrotus.By people, the research of Pharmaceutical Polysaccharides mechanism of action is found, separated the antitumor physiologically active that waits of the polysaccharide performance that obtains from natural product, play a role as immunomodulator by polysaccharide molecule often.Polysaccharide not only can activate the panimmunity cell, simultaneously normal body cell is not had toxic side effect, thereby strengthen the clinical treatment aspect that immunomodulator that biological immune reaction or antibody generates and activate is applied to cancer and immunodeficiency disease and have huge research potential as a kind of.Correspondingly, the research range of polysaccharide also constantly enlarges, and has not only comprised the research of Separation and purification, structural analysis, has also developed into gradually the structure activity relationship of polysaccharide, mechanism of action, the aspects such as clinical application.Along with deepening continuously of research, compound of polysaccharide has attracted more and more scholars' attention, and its bioactive research has obtained develop rapidly.
Summary of the invention
The objective of the invention is to extract a kind of polysaccharide from the sexual gland of Yantai Strongylocentrotus nudus, identify its chemical structure and determine its concrete active purposes.
The present invention relates to the evaluation of SEP-1 structure, namely separate the SEP-1 that obtains from the Strongylocentrotus nudus Huang, show through monose compositional analysis result, SEP-1 is a kind of dextran, the repeat unit structure of inferring SEP-1 in the present invention by chemical analysis and spectroscopic analysis as shown in Figure 1, main chain is comprised of 1 → 4 glycosidic link, and side chain is comprised of 1 → 3 glycosidic link, and the glycosidic link residue is the α configuration.Show that SEP-1 involved in the present invention is to separate first the dextran of a kind of novel structure that obtains from the Strongylocentrotus nudus of Yantai.
Above-mentioned SEP-1 has obvious proliferation function to splenic lymphocyte when 200-800 μ g/mL, and is certain dose-dependently.SEP-1 can significantly promote Turnover of Mouse Peritoneal Macrophages to engulf the ability of toluylene red in addition, in 100-400 μ g/mL concentration range, compare significant difference (P＜0.05) with control group, and can stimulate significantly Turnover of Mouse Peritoneal Macrophages RAW264.7 propagation.
Description of drawings
Fig. 1 is the repeating unit of SEP-1
Fig. 2 is SEP-1 separation and Extraction schema
Fig. 3 is SEP-1 high performance liquid phase (HPLC) color atlas
Fig. 4 is SEP-1 hydrolysate thin-layer chromatogram
Fig. 5 is gas phase (GC) color atlas of the alditol acetate of SEP-1
Fig. 6 is NaIO
4The concentration of solution-light absorption value typical curve
Fig. 7 is the gas chromatogram of the Smith degraded product of SEP-1
Fig. 8 is the infrared spectrum after the SEP-1 exhaustive methylation
Fig. 9 is gas phase (GC) color atlas in the alditol acetate GC-MS on-line analysis of SEP-1 of exhaustive methylation
Figure 10 is gas phase (GC) color atlas of the alditol acetate after the acid hydrolysis of SEP-1 part
Figure 11 is the SEP-1 UV-VIS
Figure 12 is SEP-1 infrared spectroscopy collection of illustrative plates
Figure 13 is SEP-1
1The HNMR collection of illustrative plates
Figure 14 is SEP-1
13The CNMR collection of illustrative plates
Figure 15 is that SEP-1 is to the proliferation function figure of mouse spleen lymphocyte
Figure 16 is that SEP-1 is to mouse macrophage RAW264.7 proliferation function figure
Figure 17 is that SEP-1 is engulfed the figure that affects of toluylene red on mouse macrophage RAW264.7
Embodiment
One, extract flow process
Fresh sea urchin is cut, carefully peel off gonad of Hemicentrotus seu Strongylocentrotus, filtered through gauze is removed the moisture in gonad of Hemicentrotus seu Strongylocentrotus.Add isopyknic acetone in the 500g gonad of Hemicentrotus seu Strongylocentrotus of collecting, stir and to make it fully to contact with acetone, standing 30min abandons upper strata acetone, repeatedly for several times substantially colourless to the acetone layer till, at last the acetone decompression is volatilized, get SEP-1 acetone powder 200g.The gonad of Hemicentrotus seu Strongylocentrotus acetone powder is placed in 2L distilled water, and 90 ℃ were extracted 5 hours, and 3 times repeatedly, get water extraction liquid 6L altogether, 50 ℃ are evaporated to 1.2L.
Add papoid 8g in concentrated solution, 60 ℃ of water-baths 20 hours.Utilize the Sevage method except albumen, 10-15 time.Add 0.3L Sevag reagent (chloroform: water-saturated n-butanol=5: 1), thermal agitation 15min, the centrifugal 15min of 4000rpm gets supernatant, repeats aforesaid operations 10 times.55 ℃ of concentrating under reduced pressure are removed organic reagent and liquor capacity are concentrated into 500mL.
To except slowly adding 2.5L to be chilled in advance the dehydrated alcohol of 4 ℃ in the concentrated solution of Deproteinization, and at 4 ℃ of lower hold over night precipitation polysaccharide, 5000rpm, 10min centrifugal collecting precipitation, the Crude polysaccharides that obtains with the absolute ethanol washing precipitation 3 times.Crude polysaccharides is dissolved in 50mL distilled water, obtains Crude polysaccharides 17.5g after freeze-drying.
Get the aqueous solution that Crude polysaccharides 500mg is made into 50mg/mL, cross after 0.45 μ m filter membrane filter through DEAE-52 cellulose ion exchange column (3.5cm * 25cm) separate.The distilled water wash-out, flow velocity 0.5mL/min, every pipe 5mL fraction collection, the phenolsulfuric acid method is measured sugared content by pipe, draws elution curve, collects the 8th～17 pipe, merges postlyophilization and obtains 315mg polysaccharide CP.Get polysaccharide CP30mg, be dissolved in 1mL (0.05mol/L) NaCl solution, after 0.22 μ m membrane filtration, through Sephacryl S-400 column chromatography (1.6cm * 80cm) separate, 0.05mol/L the NaCl eluant solution, flow velocity 0.5mL/min, every pipe 3mL fraction collection, sulfuric acid-phynol method detects sugared content by pipe, draw elution curve, collect the 10th～20 pipe, after the dialysis tubing dialysis desalination with molecular weight cut-off 10kD, lyophilize gets SEP-1 14mg.The leaching process schema as shown in Figure 2.
Two, property testing
(HPLC) the analysis showed that through high performance liquid phase, and the purity of SEP-1 is for being 98.501%, as shown in Figure 3.
The analysis showed that through efficient gel column chromatography (HPGPC) molecular weight of SEP-1 is 6.78 * 10
5Da.
The specific optical rotation that the WZZ-2B automatic polarimeter is measured SEP-1 is
Results of elemental analyses shows, SEP-1 does not contain N, and the quality percentage composition of C and H is respectively 39.12% and 6.68%, belongs to the hydrocarbon compositing range of neutral sugar.
SEP-1 and phenolsulfuric acid, By Anthrone Sulphuric acid and fehling reagent reaction are positive, and show that it is the reductibility polysaccharide; Be negative with the IKI reaction, show that its space structure is different from starch; Be negative with carbazole-sulfuric acid reaction, show not contain uronic acid in structure.
Three, determination of chemical structure:
One) monose compositional analysis
1, Microcellulose TLC analysis: the SEP-1 10mg that gets in the present invention is placed in ampoul tube, adds 2mL2.0mol/L trifluoroacetic acid (TFA), fills N
2Tube sealing is in 110 ℃ of hydrolysis 2h.After complete hydrolysis, 40 ℃ of concentrating under reduced pressure are removed TFA, add methyl alcohol 2.0mL evaporate to dryness, repeat 3 times to remove TFA fully.Sample is dissolved in 0.5mL distilled water, take glucose, rhamnosyl, glucuronic acid, semi-lactosi as reference substance, make tlc analysis with the contained monose kind of test sample at the Microcrystalline Cellulose plate, the developping agent that uses is: ethyl acetate: pyridine: water: acetic acid=5: 5: 3: 1, developer is aniline-phthalic acid, 110 ℃ of colour developing 10min form to determine its monose.Result hydrolysate as can be seen from Figure 4 only has a brown spot, and its R
fBe worth unanimously with dextrose standard sample, show that the SEP-1 in the present invention may be a kind of polysaccharide that is comprised of glucose.
2, gas chromatographic analysis: after the SEP-1 in the present invention is hydrolyzed into monose, be reduced into corresponding sugar alcohol under the effect of sodium borohydride, through acetylize, obtain corresponding alditol acetate derivative.By gas chromatographic analysis, the hydrolysate of SEP-1 only contains a main peak, its retention time be 19.743min as shown in Figure 5, show that SEP-1 is comprised of a kind of monose.Dextrose standard sample carries out parallel running, and the dextrose standard sample appearance time is 19.800min, and is consistent with the retention time of main peak in the GC collection of illustrative plates of alditol acetate, illustrates that SEP-1 is comprised of glucose fully.In conjunction with above-mentioned thin layer chromatography result, prove that the SEP-1 in the present invention is dextran.
Two) NaIO
4Oxidation Analysis
1, NaIO
4The drafting of solution typical curve: the NaIO that accurately prepares 10mM, 8mM, 6mM, 4mM, 2mM with redistilled water
4Solution, all dilute 250 times after take redistilled water as contrast, measure it at the light absorption value at 223nm place, draw light absorption value A
223-concentration C typical curve is seen Fig. 6.
2, take SEP-1 10mg in the present invention, be placed in brown reaction flask, add the NaIO of 30mL10mM
4, vibration makes its dissolving, jumps a queue to be placed in the dark place's oxidation of 4 ℃ of refrigerators, and interrupted oscillation, extract reaction solution 200 μ L respectively at 24h, 48h, 72h, 96h, 120h interval, dilute 250 times and measure its light absorption values at 223nm place (distilled water zeroing), according to typical curve calculating NaIO
4Consumption.Reach when stablizing until light absorption value, add 2mL ethylene glycol and stir 30min to reduce unreacted NaIO in system
4Get the solution after 2.0mL reaction finishes, make indicator with phenolphthalein, carry out titration, the burst size of calculating formic acid with the NaOH solution of 0.05mM.According to NaIO
4Consumption and the burst size of the formic acid mol ratio of calculating the different sugar glycosidic bond.
SEP-1 is through the NaIO of 10mM
4Oxidation 96h reaches balance, IO in reaction
4 -Consumption be 2.127mmol, the formic acid burst size is about 0.215mmol.Can know by inference according to principle, 1 → 4 or (with) 1 → 2 glycosidic link and 1 → 6 glycosidic link or (with) ratio of non-reduced end is 9.4: 1.
Three) Smith degraded
1, get the 10mg SEP-1, be placed in brown reaction flask, add the NaIO of 30mL10mM
4, vibration makes its dissolving, jump a queue be placed in 4 ℃ of refrigerators dark places complete oxidation after, add 2mL ethylene glycol and stir 30min to make reaction terminating.Reaction solution was to flowing water dialysis 2 days, and redistilled water was dialysed 1 day.40 ℃ of liquid in bag are evaporated to 10mL, add 30mgNaBH
4, stirring reaction 24h in dark place under room temperature, then transfer PH to 5 with 0.1M acetic acid, use successively flowing water, distill water dialysis to thoroughly desalination, lyophilize gets polysaccharide polyol.
2, above-mentioned polysaccharide polyol is placed in ampoule, adds 2mL2.0mol/L trifluoroacetic acid (TFA), fill N
2Tube sealing is in 110 ℃ of hydrolysis 2h.After complete hydrolysis, 40 ℃ of concentrating under reduced pressure are removed TFA, add methyl alcohol 2.0mL evaporate to dryness, repeat 3 times to remove TFA fully.Sample after hydrolysis is dissolved in 3mL distilled water, adds 25mg NaBH
4, reductase 12 h under room temperature.After reaction is completed, destroy excessive NaBH with Glacial acetic acid
4, the pH value to 5 of regulator solution, evaporated under reduced pressure adds 3mL methyl alcohol and a Glacial acetic acid, then evaporated under reduced pressure, repeats 3 times postlyophilization.
3, the sample after freeze-drying is added in the 2mL anhydrous pyridine, ultrasonic 15min adds the 1mL acetic anhydride after it is dissolved fully, fills N
2Rear close plug, 100 ℃ of reaction 2h.Reaction is cooled to room temperature after finishing, and adds the 5mL distilled water, regulates pH to neutral with HCl.Reaction solution extracts repeatedly with chloroform, until chloroform layer is colourless, merge the chloroform phase of collecting, with equal-volume distilled water wash 3 times, chloroform layer anhydrous sodium sulphate standing and drying 10min, refilter and remove the sodium sulfate solid, chloroform is concentrated into the laggard promoting the circulation of qi phase of 0.1mL chromatogram (GC) analysis, as shown in Figure 7.
Gas chromatograph results shows in the Smith degraded product of SEP-1 glycerine (retention time is 4.106min), tetrahydroxybutane (retention time is 8.034min) and glucose (retention time is 23.590min), and the ratio that draws the three after peak area normalization method is 1: 8.8: 5.3.Analysis-by-synthesis Smith degraded and NaIO
4The result of oxidation obtains: 1 → 3 glycosidic link in the SEP-1 elaboration, the mol ratio of 1 → 4 glycosidic link and 1 → 6 glycosidic link and non-reduced end is 5.5: 9: 1.
Four) methylation analysis
1, the SEP-1 10mg that takes in the present invention is placed in reaction flask, vacuum-drying 6h.The DMSO that dried sample adds 4mL to process with the 4A molecular sieve, the room temperature magnetic agitation is until the polysaccharide sample dissolves rear at N fully
2The NaOH powder art 20mg that adds the drying of grinding under protection, stirring at room 3h, then ice bath 5min, after the solution in the question response bottle is fully freezing, dropwise add the 0.5mL methyl iodide, continues magnetic agitation reaction 3h, and the room temperature underpressure distillation eliminates excessive methyl iodide.
2, repeatedly extract with chloroform after adding the 10mL distilled water in the reaction solution, until chloroform layer is colourless, merge the chloroform phase of collecting, with equal-volume distilled water wash 3 times, chloroform layer removes by filter the sodium sulfate solid after with anhydrous sodium sulphate standing and drying 10min, the chloroform that obtains reduces pressure mutually and is evaporated to driedly, the methylate that obtains is dissolved in lyophilize in 1mL distilled water, then vacuum-drying 5h.After repetition aforesaid operations 3 times, the sample that takes a morsel carries out infrared detection.The 3300cm of raw sample on the IR spectrum
-1Locate strong and wide hydroxyl peak and disappear, and 2900cm
-1When the methyl peak at place strengthened relatively, as shown in Figure 8, interpret sample was by exhaustive methylation.
3, add the TFA of 4mL2mol/L in the sample after the exhaustive methylation, seal under rear 110 ℃ and be hydrolyzed 4h, the solution decompression evaporate to dryness in reaction flask, then add 3mL methyl alcohol, evaporate to dryness repeats 3 times to eliminate excessive TFA.Sample after hydrolysis is dissolved in 3mL distilled water, adds 25mg NaBH
4, reductase 12 h under room temperature, then with the pH value to 5 of Glacial acetic acid regulator solution, evaporated under reduced pressure adds 3mL methyl alcohol and a Glacial acetic acid, then evaporated under reduced pressure, repeats 3 times, then vacuum-drying 4h.
4, dried sample adds the 2mL anhydrous pyridine, adds the 1mL acetic anhydride after ultrasonic 5min dissolves fully, fills N
2Rear close plug, 100 ℃ of reaction 2h.Reaction is cooled to room temperature after finishing, and adds the 3mL distilled water, regulates pH to neutral with HCl.Reaction solution extracts repeatedly with chloroform, until chloroform layer is colourless, merge the chloroform phase of collecting, use equal-volume distilled water wash 3 times, chloroform layer removes by filter the sodium sulfate solid after with anhydrous sodium sulphate standing and drying 10min, and chloroform is concentrated into the laggard promoting the circulation of qi matter of 0.1mL combination analysis.
Four derivative quasi-molecular ions that methylate have appearred in the SEP-1 methylate in GC, as shown in Figure 9, its retention time is respectively 12.269min, 13.725min, 14.456min and 14.756min.each mass spectrum of online information retrieval, preliminary definite each mass spectral:mass spectrographic ownership, according to elementary fragment and the secondary fragment in the cracking rule ownership mass spectrum of Partially methylated sugars alcohol acetic ester derivative, by resolving, in the GC spectrogram, four compositions are followed successively by: 1, 5-two-oxy-acetyl-2, 3, 4, 6-four-oxygen-methyl-glucose (2, 3, 4, 6-Me4-Glc), 1, 4, 5-three-oxy-acetyl-2, 3, 6-three-oxygen-methyl-glucose (2, 3, 6-Me3-Glc), 1, 3, 5-three-oxy-acetyl-2, 4, 6-three-oxygen-methyl-glucose (2, 4, 6-Me3-Glc) He 1, 3, 4, 6-four-oxy-acetyl-2, 3-two-oxygen-methyl-glucose (2, 3-Me2-Glc) four component mol ratios are about 1: 9: 5: 1.Comprehensive Smith degraded and NaIO
4Result as can be known, SEP-1 be a kind of contain non-reduced end 1 →, the dextran of the composition of 1 → 4 and 1 → 3 glycosidic link, three's ratio is about 1: 9: 5.
Five) part acid hydrolysis
Get the SEP-1 10mg in the present invention, be placed in ampoul tube, add the TFA5mL of 0.3mol/L, fill N
2Tube sealing, 100 ℃ of hydrolysis 18h use in NaOH and unnecessary TFA, dialysis, lyophilize gets hydrolysate, then through NaIO
4Oxidation and NaBH
4Get corresponding sugar alcohol after reduction, sugar alcohol only detects tetrahydroxybutane through hydrolysis and the laggard promoting the circulation of qi analysis of hplc of acetylize, as shown in figure 10, shows that the main chain of SEP-1 is comprised of 1 → 4 glycosidic link.
Six) ultraviolet spectral analysis
SEP-1 1mg in the present invention is made into the 1.0mg/mL aqueous solution, scan in the scope of 200～600nm with ultraviolet spectrophotometer, see Figure 11, it all without charateristic avsorption band, shows that the SEP-1 polysaccharide is without protein and nucleic acid at 260nm and 280nm place.
Seven) Infrared spectroscopy
SEP-1 in the present invention is got 1.0mg with the KBr compressing tablet, at 4000～400cm after vacuum-drying 12h
-1Scope in carry out Infrared spectrum scanning, result can be found out: as shown in figure 12 at 3417.7cm
-1A stronger broad peak appears in the place, is the stretching vibration of-OH; At 2947.6cm
-1And 2842.5cm
-12 stretching vibrations that absorption peak is C-H, 1378.7cm
-1The peak at place is the angle vibration of C-H, these three groups of charateristic avsorption bands that the peak is polysaccharide.1020.6cm
-1The chief component monose of the strong absorption explanation SEP at place is glucose, at 969.7cm
-1The absorption peak and 1020.6 at place, 1032.2 and 1156.0cm
-1The absorption peak at place shows that SEP-1 is comprised of α-D-Glucopyranose.
Eight) nucleus magnetic resonance (NMR) is analyzed
Get the SEP-1 30mg in the present invention, be dissolved in the D of 0.5mL after at room temperature oil pump vacuum-drying 3h
2In O, measure it on the AVANCE500 nuclear magnetic resonance analyser
1H NMR spectrum; Separately get cryodesiccated SEP-1 200mg at the BRUKER AVANCE III400MHz of company wide chamber solid-state nuclear magnetic resonance measurements
13C NMR spectrum.As shown in figure 13, exist
1In H NMR, there are two anomeric proton signals the low place of δ＞4.5ppm, and its chemical shift is respectively δ 5.35ppm and δ 5.39ppm, points out this polysaccharide to contain two kinds of α glycosidic links, and this signal proportion is different, and two kinds of glycosidic link skewness weighing apparatuses are described.Scheme as shown in figure 14,
13In C NMR, two fignal centers appear in the low place of δ 90～110ppm, are the fignal center of anomeric carbon, and its chemical shift is respectively δ 102.72 and 98.26ppm, with
1In H NMR spectrum, two anomer hydrogen fignal centers are corresponding.
The mode of connection that contains 1 → 4 and 1 → 3 two kind of glycosidic link in the basic repeating unit of its presentation of results composition SEP-1, and saccharide residue is configured as the α type.The result that obtains with chemical analysis methods such as methylation analysis, periodate oxidation, Smith degraded and part acid hydrolysiss is consistent.
Four, comprehensive analysis described above can get following result:
1, monose compositional analysis result shows: the SEP-1 in the present invention is dextran.
2, periodate oxidation, Smith degraded and the result that methylates show the SEP-1 in the present invention be a kind of contain non-reduced end 1 →, by the dextran that forms of 1 → 3 and 1 → 4 glycosidic link, its ratio is about 1: 5: 9.
3, the acid-hydrolyzed result of part shows that the glucose mode of connection that consists of the SEP-1 main chain is α-Isosorbide-5-Nitrae glycosidic link.
4, the NMR analytical results shows and contains 1 → 3 and 1 → 4 two kind of glycosidic link in the basic repeating unit that forms SEP-1, and saccharide residue is configured as the α type.
Therefore, the structure of the basic repeating unit of the SEP-1 in the present invention is as follows, and it is a kind of novel texture polysaccharide of finding from gonad of Hemicentrotus seu Strongylocentrotus.
Material and reagent: SEP-1 (self-control, detecting through HPLC is homogeneous polysaccharide), LPS (Sigma), CCK-8 (Sigma), MTT (Sigma), RPMI Medium1640 (Gibco), splenic lymphocyte parting liquid (folium ilicis chinensis biomaterial company limited)
Instrument Bechtop (Suzhou Decontamination Equipment Plant), CO
2Incubator (U.S. ThermoForma company), Mode1680 type enzyme-linked immunosorbent assay instrument (U.S. Bio-Rad company)
One, the impact of SEP-1 on mice spleen lymphocytes proliferation
Get 6-8 ICR mouse in age in week, the cervical vertebra dislocation is put to death, and aseptic preparation splenic lymphocyte suspension is adjusted mouse spleen lymphocyte concentration to 2 * 10 with the RPMI1640 that adds calf serum without phenol red medium
6/ mL, add 100/ μ L cell suspension in 96 orifice plate every holes, then add the nutrient solution of the SEP-1 of different concns, final concentration is respectively 6.25,12.5,25,50,100,200,400 μ g/mL, every Kongzui final volume is 200 μ L, sets simultaneously blank hole and positive controls (LPS50 μ g/mL).All establish 4 multiple holes, put 37 ℃, 5%CO for every group
2After cultivating 36h in incubator, every hole adds CCK-820 μ L, continues to cultivate 1h, and microplate reader 570nm detects the OD value in the place.Result shows that SEP-1 has obvious proliferation function to splenic lymphocyte as shown in figure 15, and is dose-dependently when 200-800 μ g/mL.
Two, the impact of SEP-1 on the peritoneal macrophage RAW264.7 of mouse
The impact of 1 SEP-1 on normal RAW264.7 cell-proliferation activity
To add density be 1.0 * 10 in every hole in 96 orifice plates
5The RAW264.7 cell 100 μ L of/mL add the nutrient solution of the SEP-1 of different concns after 6h is adherent, final concentration is respectively 0,25,125,250,500,1000 μ g/mL, every Kongzui final volume is 200 μ L, sets simultaneously blank group, positive controls (LPS50 μ g/mL).After hatching 24h, the MTT20 μ L that every hole adds concentration 5mg/mL puts 37 ℃, 5%CO
2Incubator is abandoned supernatant after cultivating 4h, and every hole adds DMSO150 μ L, shake well 10min, and the place measures absorbancy at microplate reader 570nm wavelength.Result as shown in figure 16, SEP-1 can the remarkable propagation that must promote Turnover of Mouse Peritoneal Macrophages when high density.
The impact of 2 SEP-1s on normal RAW264.7 cytophagy toluylene red function
To add density be 1.0 * 10 in every hole in 96 orifice plates
5The RAW264.7 cell 100 μ L of/mL add the SEP-1 nutrient solution of different concns after 12h is adherent, final concentration is respectively 0,25,50,100,200,400 μ g/mL, every Kongzui final volume is 200 μ L, sets simultaneously positive control (LPS50 μ g/mL).Each concentration is established 4 multiple holes.Be placed in 37 ℃, 5%CO
2After cultivating 24h in incubator, abandon supernatant, PBS washing 2 times, every hole adds the 0.1% toluylene red physiological saline of 100 μ L, puts 37 ℃, 5%CO
2Incubator is abandoned supernatant after cultivating 1h, and every hole adds PBS liquid 200 μ L washing 3 times, and every hole adds lysis liquid 150 μ L, and after 37 ℃ of lysing cell 3h, the place measures absorbancy at microplate reader 550nm wavelength.Result illustrates that SEP-1 can significantly strengthen macrophage phagocytic toluylene red ability as shown in figure 17.
Therefore, can find out that from embodiment 2 SEP-1 the present invention is a kind of immunostimulant, can strengthen significantly immunocompetence, and be undertaken by different immunomodulatory modes, for follow-up anti-tumor experiment is laid a good foundation.
Claims (6)
1. SEP-1 with α-Isosorbide-5-Nitrae-dextran main chain is characterized in that containing 1 → 4 and 1 → 3 two kind of glycosidic link in the repeated structural unit of described polysaccharide, wherein main chain is 1 → 4 glycosidic link, side chain is comprised of 1 → 3 glycosidic link, and saccharide residue is the α configuration, and its relative molecular weight is 6.78 * 10
5Da, specific optical rotation
It is for separating first the dextran of a kind of novel structure that obtains from the Strongylocentrotus nudus of Yantai.
2. SEP-1 claimed in claim 1 has main chain and is comprised of α-Isosorbide-5-Nitrae-glycosidic link, and side chain is by α-1, the repeat unit structure that the 3-glycosidic link forms, and described repeated structural unit structure is as follows.
3. the preparation method of SEP-1 according to claim 1 is characterized in that realizing as follows:
(1) fresh sea urchin is cut, carefully peel off gonad of Hemicentrotus seu Strongylocentrotus, filtered through gauze is removed the moisture in gonad of Hemicentrotus seu Strongylocentrotus, adds isopyknic acetone to carry out skimming treatment in gonad of Hemicentrotus seu Strongylocentrotus, gets the SEP-1 acetone powder; The gonad of Hemicentrotus seu Strongylocentrotus acetone powder uses respectively papoid and Sevage method to remove protein in extracting solution after 90 ℃ of hot water extract repeatedly, and organic reagent and concentrated extracting solution are removed in 55 ℃ of underpressure distillation;
(2) dehydrated alcohol that slowly adds 4 times of volumes to be chilled in advance 4 ℃ in the above-mentioned extracting solution, and at 4 ℃ of lower hold over night precipitation polysaccharide, 5000rpm, the 10min centrifugal collecting precipitation, with absolute ethanol washing precipitation 3 times, will precipitate and again be dissolved in the hot water postlyophilization, obtain the gonad of Hemicentrotus seu Strongylocentrotus Crude polysaccharides;
(3) above-mentioned Crude polysaccharides is made into the aqueous solution of 50mg/mL, separate through DEAE-52 cellulose ion exchange column after crossing 0.45 μ m filter membrane filter, the distilled water wash-out, flow velocity 0.5mL/min, every pipe 5mL fraction collection, the phenolsulfuric acid method is measured sugared content by pipe, draws elution curve, collect the 8th～17 pipe, merge postlyophilization and obtain polysaccharide CP;
(4) get above-mentioned polysaccharide CP30mg, be dissolved in 1mL (0.05mol/L) NaCl solution, after 0.22 μ m membrane filtration, through Sephacryl S-400 column chromatography for separation, 0.05mol/L the NaCl eluant solution, flow velocity 0.5mL/min, every pipe 3mL fraction collection, sulfuric acid-phynol method detects sugared content by pipe, draw elution curve, collect the 10th～20 pipe, after the dialysis desalination, lyophilize gets SEP-1.
4. the purposes of the described SEP-1 of claim 1.
5. purposes claimed in claim 4, be included in the application in preparation immunomodulator and antitumour auxiliary drug.
6. claim 5 is described, the application of priority protection in the preparation immunomodulator.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497162A (en) * | 2015-01-05 | 2015-04-08 | 中国药科大学 | Urchin yellow polysaccharide with liver protecting function and application thereof |
| CN109406702A (en) * | 2018-10-12 | 2019-03-01 | 山东省科学院生物研究所 | A kind of method of the monosaccharide residue regularity of distribution in analysis polysaccharide structures |
| CN110894244A (en) * | 2019-10-17 | 2020-03-20 | 中国药科大学 | Structure of ground beetle polysaccharide and application thereof |
| CN112390898A (en) * | 2019-08-18 | 2021-02-23 | 于荣敏 | Arca inflata reeve immunoregulation and anti-tumor polysaccharide and preparation method and application thereof |
| CN113943381A (en) * | 2021-11-29 | 2022-01-18 | 滨州医学院 | Novel purification method, molecular structure and application of sea urchin gonadal polysaccharide |
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| CN102134284A (en) * | 2011-04-12 | 2011-07-27 | 中国药科大学 | Anti-hepatoma drug strongylocentrotus nudus egg polysaccharide SEP-1 based on immune regulation |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497162A (en) * | 2015-01-05 | 2015-04-08 | 中国药科大学 | Urchin yellow polysaccharide with liver protecting function and application thereof |
| CN109406702A (en) * | 2018-10-12 | 2019-03-01 | 山东省科学院生物研究所 | A kind of method of the monosaccharide residue regularity of distribution in analysis polysaccharide structures |
| CN109406702B (en) * | 2018-10-12 | 2021-07-09 | 山东省科学院生物研究所 | Method for analyzing distribution rule of monosaccharide residues in polysaccharide structure |
| CN112390898A (en) * | 2019-08-18 | 2021-02-23 | 于荣敏 | Arca inflata reeve immunoregulation and anti-tumor polysaccharide and preparation method and application thereof |
| CN110894244A (en) * | 2019-10-17 | 2020-03-20 | 中国药科大学 | Structure of ground beetle polysaccharide and application thereof |
| CN110894244B (en) * | 2019-10-17 | 2021-06-11 | 中国药科大学 | Structure of ground beetle polysaccharide and application thereof |
| CN113943381A (en) * | 2021-11-29 | 2022-01-18 | 滨州医学院 | Novel purification method, molecular structure and application of sea urchin gonadal polysaccharide |
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| CN103382229B (en) | 2016-01-13 |
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